PCR Analysis

Overview

Depending on the information desired, there are many different methods to analyze the products of a PCR reaction. Agarose gel electrophoresis is a common technique to detect the presence or absence of the target sequence and the length of the fragment. Mutation detection methods such as denaturing gradient gel electrophoresis (DGGE) and temporal temperature gel electrophoresis (TTGE) use an acrylamide gel to assist with identifying mutations in the PCR product. Semiquantitative analysis techniques enable agarose gel electrophoresis to be used to approximate the starting quantity of material. Sequencing and next-generation sequencing technique are used when the sequence of the amplicon must be determined.

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Gel Electrophoresis

PCR products are most commonly analyzed by agarose gel electrophoresis. The results can be visualized by ethidium bromide or non-toxic dyes such as SYBR® green. The intensity of the band can be used to estimate the amount of product of given molecular weight relative to a ladder. Gel electrophoresis also shows the specificity of the reaction, where the presence of multiple bands indicates secondary amplification products.

Semiquantitative Analysis

PCR and reverse transcriptase PCR (RT-PCR) are commonly used methods for detecting species of DNA and RNA, respectively. However the quantitative application of these methods has been limited. Theoretically, PCR amplifies template DNA exponentially, with a doubling of template every cycle, so that relative differences between samples can be measured as the intensity of bands on the gel.

The limitation is that in later phases of PCR the efficiency decreases and the amplification reaches a plateau. This means that the amount of product in the reaction is no longer proportional to the amount of starting material and cannot be quantified. Relative amounts of amplified DNA in different PCR reactions can only be compared when the reactions are still in the exponential phase of amplifications. For these reasons, qPCR is the most commonly used method for quantitative analysis.

If proper care is taken to ensure that saturation has not been reached, end-point PCR can be successfully used as a semiquantitative measure of relative differences in template input between samples (see Bio-Rad bulletin 2915).To ensure that the reaction is within the exponential phase of amplification, samples can be amplified with increasing numbers of cycles, run on gel electrophoresis, and quantitated with a gel imager and software. Specific techniques for semiquantitative PCR include relative RT-PCR and competitive RT-PCR.

In relative RT-PCR, two primer pairs are designed for a multiplexed PCR reaction — one for the gene of interest and another for a housekeeping gene (Marone et al. 2001). The primer pairs must be designed not to form dimers and to form products of sufficiently different lengths that they can be distinguished via gel electrophoresis. Pilot experiments must be run to check the product after different numbers of cycles and to determine the exponential phase of the reaction for both primer sets. Using gel imaging software to determine the band intensity, the relative fold change can then be calculated between the samples normalized to the housekeeping gene. The challenge with this approach is that housekeeping genes needed for normalization typically have much higher expression than the gene of interest. One approach to this issue is to use lower-expressing housekeeping genes and to use higher concentrations of primers for the gene of interest, and lower concentrations of primers for the housekeeping gene, to favor formation of the lower-expressing product.

In competitive RT-PCR, an exogeneous RNA or DNA control is synthesized and spiked into the samples (Francesco et al. 1993). The control must be designed so that it contains the primer binding sequences as the target but will yield a product of a different length. The control is then diluted into a standard curve of known concentration, and spiked into replicates of the sample. The exogeneous control and the sample compete for primers and other reaction components and amplify into two distinct bands. Comparing the intensity of the band for the sample to the control of known quantity yields a semiquantitative measure of the amount of the transcript in the sample.

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